Wagener C, Clark B R, Rickard K J, Shively J E
J Immunol. 1983 May;130(5):2302-7.
A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.
描述了一种测定单克隆抗体亲和力常数和抗原交叉反应性的通用方法。该方法在均相竞争放射免疫分析中使用生物素标记的抗体、放射性标记的抗原和抗生物素蛋白作为沉淀剂。这种方法消除了在使用单克隆抗体的免疫分析中经常遇到的抗原 - 抗体复合物沉淀不完全或变化的问题。使用该检测系统，我们能够快速测定多种针对癌胚抗原（CEA）产生的单克隆抗体的亲和力常数。在前一篇论文中表明，五种单克隆抗体识别CEA上不同的表位。在使用这五种单克隆抗体的抗原结合实验中，在抗体过量时结合的放射性标记CEA的百分比范围为30%至92%。CEA交叉反应抗原、正常交叉反应抗原（NCA）和肿瘤提取的CEA相关抗原（TEX）被五种抗体中的一种显著结合，另外两种抗体结合程度较小。两种抗体不结合大量的NCA或TEX。在抑制研究中，导致125I标记的CEA结合被50%抑制的未标记CEA的量为每管3.7至760 ng。对于两种抗体，显示相同程度抑制的TEX量比CEA量高23倍，对于第三种抗体则比CEA量高351倍。CEA的亲和力常数范围为1.0×10(8)至5.1×10(10) M-1。针对其中一种抗体测定的NCA和TEX的亲和力常数与CEA相比低三个数量级。如一种单克隆抗体结合的低比例所示，放射性标记CEA的异质性很可能是放射性碘化损伤导致的假象。本报告中描述的方法的应用应消除在测定单克隆抗体亲和力常数或在竞争性均相免疫分析中使用单克隆抗体时最常遇到的问题。
